Eluting matrix and uses thereof

ABSTRACT

CXCL12 polypeptide eluting matrices encapsulating at least one cell are described for use in the treatment of autoimmune disorders.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of and claims priority to U.S. patentapplication Ser. No. 15/019,449, filed Feb. 9, 2016, which is acontinuation application of and claims priority to International PatentApplication No. PCT/US2013/068916, filed Nov. 7, 2013, the entirecontents of which of each are incorporated herein by reference.

STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING

A Sequence Listing in ASCII text format, submitted under 37 C.F.R.§1.821, entitled 1416-4CT3_ST25.txt, 6,129 bytes in size, generated onMay 8, 2017 and filed via EFS-Web, is provided in lieu of a paper copy.The Sequence Listing is incorporated herein by reference into thespecification for its disclosures.

BACKGROUND OF THE INVENTION

Transplantation is a potentially curative approach for individuals withautoimmune disorders such as type I diabetes mellitus (T1DM), but itsutility is limited by acute and chronic immune rejection of transplantedcells (N. Papeta et al. Transplantation 83, 174 (Jan. 27, 2007); A. M.Shapiro et al. The New England journal of medicine 355, 1318 (Sep. 28,2006); J. S. Kaddis et al. JAMA 301, 1580 (Apr. 15, 2009); R. P.Robertson. The New England journal of medicine 350, 694 (Feb. 12, 2004);R. B. Jalili et al. Diabetes 59, 2219 (September, 2010); and V.Vaithilingam, The review of diabetic studies: 7, 62 (Spring, 2010)).Immune rejection is currently managed by continuous systemic immunesuppression, an approach that has not shown significant long-termeffectiveness, while exposing recipients to increased risks of infectionand cancer (A. G. Mallett, G. S. Korbutt. Tissue engineering. Part A 15,1301 (June, 2009); N. Sakata et al. World journal of gastrointestinalpathophysiology 3, 19 (Feb. 15, 2012); M. C. Poznansky et al. TheJournal of clinical investigation 109, 1101 (April, 2002); and M. C.Poznansky et al. Nature medicine 6, 543 (May, 2000)). Alternativetherapies that can overcome the need for systemic immunosuppressionthrough the induction of local anatomic site specific immune modulationwould be desirable.

SUMMARY OF THE INVENTION

CXCL12 polypeptides can repel effector T-cells while recruitingimmune-suppressive regulatory T-cells to an anatomic site. It has nowbeen determined that CXCL12 is capable of overcoming both acute andchronic immune destruction of an implanted matrix in a site specificmanner, abrogating the need for concurrent systemic immune suppression.

In one aspect, provided herein relates to compositions comprising atleast one cell encapsulated in a CXCL12 polypeptide eluting matrix.

In some embodiments, the CXCL12 polypeptide eluting matrix can becharacterized by a release of the CXCL12 polypeptide at a ratesufficient to repel effector T cells. For example, in some embodiments,the CXCL12 polypeptide eluting matrix can be characterized by a releaseof the CXCL12 polypeptide at a rate of at least about 1.0 ng/mL/hr. Insome embodiments, the CXCL12 polypeptide can be released from the CXCL12polypeptide eluting matrix at a rate of at least about 1.5 ng/mL/hr, atleast about 2 ng/mL/hr, at least about 2.5 ng/mL/hr, at least about 3ng/mL/hr, at least about 4 ng/mL/hr, at least about 5 ng/mL/hr orhigher. In some embodiments, the CXCL12 polypeptide can be released at arate of about 1.0 ng/mL/hr to about 3 ng/mL/hr. In some embodiments, theCXCL12 polypeptide can be released at a rate of about 1.75 ng/ml/hr. Byway of example only, the composition described herein can becharacterized by an ability of repelling effector T cells in vitro in aboyden chamber.

The CXCL12 polypeptide can be present in the eluting matrix at anyconcentration. In some embodiments, the concentration of the CXCL12polypeptide can be optimized, e.g., for a desired release rate of theCXCL12 polypeptide from the eluting matrix and/or its release duration.In some embodiments, the CXCL12 polypeptide can be present in the matrixat a concentration of about 100 ng/mL. In some embodiments, the CXCL12polypeptide can be present in the matrix at a concentration of about 100ng/mL to about 1 μg/mL. In some embodiments, the concentration of CXCL12polypeptide within the eluting matrix can be maintained at aconcentration of about 100 ng/ml to about 1 μg/ml for about 3 months toabout 2 years. In some embodiments, the concentration of the CXCL12polypeptide within the eluting matrix can be maintained at about 100ng/mL to about 1 μg/mL for about 3 months to about 2 years uponimplantation of the composition in a subject. In some embodiments, themaintained concentration of CXCL12 polypeptide within the matrix can beabout 100-200 ng/ml.

To form a CXCL12 polypeptide eluting matrix, the CXCL12 polypeptides canbe pre-loaded into the eluting matrix or produced in situ in the elutingmatrix. For example, in some embodiments, the CXCL12 polypeptides withinthe CXCL12 polypeptide eluting matrix can be provided by CXCL12polypeptide-secreting cells or cells engineered to secrete CXCL12polypeptides. In one embodiment, the CXCL12 polypeptides within thematrix can be provided by the islet cells within the matrix.

In some embodiments, the CXCL12 polypeptides present in the elutingmatrix can comprise an amino acid sequence based on the species of asubject to be treated. For example, in some embodiments, the CXCL12polypeptide can comprise a human CXCL12 polypeptide.

The CXCL12 polypeptide eluting matrix can be characterized by variousmatrix structures. For example, in some embodiments, the CXCL12polypeptide eluting matrix can form a capsule. In some embodiments, theCXCL12 polypeptide eluting matrix can be in a form of solid or foammatrix. In some embodiments, the CXCL12 polypeptide eluting matrix canform multi-compartment or multi-layered matrix. In these embodiments,the cell(s) and the CXCL12 polypeptide can be present in the same ordifferent compartments or layers of the CXCL12 polypeptide elutingmatrix.

The thickness of the CXCL12 polypeptide eluting matrix can be varied tosuit the needs of various applications. By way of example only, thethickness of the CXCL12 polypeptide eluting can be adjusted for fastrelease or slow release of the CXCL12 polypeptide from the elutingmatrix. In some embodiments, the matrix thickness can be about 200-about500 microns.

The cell(s) encapsulated in the CXCL12 polypeptide eluting matrix can becollected or derived from any source, any species, and/or any tissuetype. Additionally or alternatively, the cell(s) encapsulated in theCXCL12 polypeptide eluting matrix can be differentiated from stem cellsto specific cell types. In some embodiments, the cell(s) encapsulated inthe CXCL12 polypeptide eluting matrix can be autologous. In someembodiments, the cell(s) encapsulated in the CXCL12 polypeptide elutingmatrix can be allogeneic cell(s) or xenogeneic cell(s).

In some embodiments, the cell(s) encapsulated in the CXCL12 polypeptideeluting matrix can retain their function and/or activity for a desiredperiod of time, e.g., upon implantation of the compositions in asubject. In some embodiments, the cell(s) can retain their functionand/or activity for at least about 1 month or longer, including, e.g.,at least about 2 months, at least about 3 months or longer. In someembodiments, the cell(s) can retain their function and/or activity forat least about 1 month or longer after implantation of the compositiondescribed herein in a subject.

Depending on types of cells encapsulated in the CXCL12 polypeptideeluting matrix, the cells can perform different functions and/oractivity. In some embodiments, the cell(s) encapsulated in the CXCL12polypeptide eluting matrix can regulate blood glucose level in asubject. For example, upon implantation, the cell(s) encapsulated in theCXCL12 polypeptide eluting matrix can release insulin in response tosurrounding or ambient glucose level. In these embodiments, the cell(s)encapsulated in the CXCL12 polypeptide eluting matrix can comprise anislet cell. The islet cell can be an insulin producing cell, an isletcell derived from an induced pluripotent stem (iPS) cell, a porcineislet cell, a human islet cell, or any combinations thereof.

In some embodiments, the cell(s) encapsulated in the CXCL12 polypeptideeluting matrix can be removed. In some embodiments, the cell(s) can beprovided to the CXCL12 polypeptide eluting matrix in vivo.

The CXCL12 polypeptide eluting matrix can comprise at least one or morebiocompatible biopolymers. The biocompatible polymers can bebiodegradable or non-degradable. The biocompatible polymers can becarbohydrate-based, protein-based, and/or synthetic. In someembodiments, the biocompatible polymers can be selected such that theyare inert to encapsulated cells (e.g., no stimulation or inhibition ofcell signaling), and are permeable to the CXCL12 polypeptide to beeluted and optionally permeable to a target molecule to be sensed. Insome embodiments, the biocompatible polymers can be selected such thatthe average pore size of the eluting matrix excludes molecules that aregreater than about 130 kD.

In some embodiments, the CXCL12 polypeptide eluting matrix can comprisean alginate gel. The alginate gel can comprise mannuronic acid (M) andguluronic acid (G) at a (M/G) ratio selected to achieve propertiesspecific for individual applications. Exemplary properties of thealginate gel that can be optimized by the M/G ratio include, but are notlimited to, molecular weight cut-off, porosity, pore size, gel strength,and/or release profile of the CXCL12 polypeptide.

In some embodiments, the alginate gel can comprise a high mannuronicacid content. In some embodiments, the alginate gel can comprisemannuronic acid (M) and guluronic acid (G) at a (M/G) ratio of about 1or greater than 1. In some embodiments, the concentration of thealginate gel can vary from about 1% w/v to about 5% w/v. In someembodiments, the concentration of the alginate gel can be about 2% w/v.

In some embodiments, the composition described herein can furthercomprise a layer of cells that can express the CXCL12 polypeptide. Insome embodiments, the CXCL12 polypeptide-expressing cells can comprisemesothelial cells.

In some embodiments, the composition described herein can furthercomprise an absorbable layer of a CXCL12 polypeptide over the elutingmatrix.

In some embodiments, the compositions can be formulated to be injectablecompositions.

In various embodiments, the compositions described herein can beimplanted or injected at a target site in a subject for treatment of adisease or disorder. In some embodiments, the compositions can compriseat least one islet cell encapsulated in a CXCL12 polypeptide elutingmatrix. Accordingly, in some embodiments, the composition, e.g., fortreatment of diabetes, can be characterized as a composition comprisingallograft or xenograft islet cells encapsulated in a CXCL12 polypeptideeluting matrix wherein the matrix is characterized by: (a) a matrixthickness from 200-500 microns, and a concentration of CXCL12polypeptide in the matrix from about 100 ng/ml to about 1 μg/ml; (b) aporosity such that agents regulating serum glucose concentration in asubject having type I diabetes diffuse through the matrix; and (c)insulin production by the islet cells based on the interaction of theagents with the islet cells, the insulin being released through thematrix and at a rate sufficient to regulate the serum concentration orblood level of glucose in the subject. The matrix thickness, CXCL12concentration and/or elution rate can be tuned to inhibit degradation ofthe islet cells for a period of at least or up to about 4 months,thereby providing control of the blood glucose levels in the subjectduring the period. In some embodiments, the matrix thickness, CXCL12concentration and/or elution rate can be tuned to inhibit degradation ofthe islet cells for a period of about 6 months or longer.

In another aspect, methods for providing islet cells to a subject inneed thereof are also described herein. The method comprises implantingone or more embodiments of the compositions described herein into thesubject, wherein the islet cells regulate blood glucose levels in thesubject for a period of time. By way of example only, the islet cellsencapsulated in the CXCL12 polypeptide eluting matrix, upon implantationin the subject, can regulate blood glucose levels in the subject for aperiod of at least about 1 month or longer, including, e.g., at leastabout 2 months, at least about 3 months, at least about 6 months, atleast about 9 months, at least about 1 year, at least about 2 years orlonger.

In some embodiments, the islet cells encapsulated in the CXCL12polypeptide eluting matrix can maintain or restore the fasting serumconcentration of glucose in the subject at a blood level of betweenabout 80 mg/dl and about 120 mg/dl.

In some embodiments, the CXCL12 polypeptide eluting matrix is notdegraded by effector T-cells or macrophages.

In some embodiments, regulatory T-cells can be present at the site ofimplantation. In some embodiments, effector-T cells can be absent fromthe site of implantation. By way of example only, the presence of theregulatory T-cells or the absence of the effector T-cells can bemeasured by flow cytometry or immunohistochemistry.

In some embodiments, the subject can receive repeated implantation of acomposition comprising at least one islet cell encapsulated in a CXCL12polypeptide eluting matrix.

A further aspect described herein provides methods for replenishingislet cells in a subject in need thereof having an existing xenograftislet cell deposit. The method comprises (a) assessing the half-life ofthe islet cells existing in the subject; (b) providing islet cells tothe subject such that the aggregated half-life of the islet cells is ata therapeutic level to provide control of the glucose levels in thesubject for a period of time; and (c) repeating steps (a) and (b) basedon the half-life of the islet cells. In some embodiments, the half-lifeof the islet cells exiting in the subject can be assessed or estimatedby monitoring changes in the blood glucose level in a subject. Forexample, a return of blood glucose to a diabetic state can be indicativeof a need to replenish islet cells in the subject.

Compositions comprising a CXCL12 polypeptide eluting matrix are alsoprovided herein. The matrix can be characterized by: (a) a porosity suchthat CXCL12 polypeptide is slowly eluted from the composition andpenetrates an active site of an autoimmune disease; (b) an agent thatrenders the composition non-migratory after administration such that asubstantial portion of the matrix remains located in and about the siteof administration; wherein the CXCL12 polypeptide concentration andelution rate are selected to inhibit further development of saidautoimmune disease. In some embodiments, the compositions describedherein can be administered by injection. In these embodiments, thecompositions can be formulated to be injectable compositions.

Other features and advantages of the invention will be apparent from thedetailed description, and from the claims. Thus, other aspects of theinvention are described in the following disclosure and are within theambit of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The following Detailed Description, given by way of example, but notintended to limit the invention to specific embodiments described, maybe understood in conjunction with the accompanying figures, incorporatedherein by reference.

FIGS. 1A-1D show that coating of alloislets with a high concentration ofCXCL12 delays rejection. FIG. 1A is a survival curve of the proportionof allograft islets after transplantation. BALB/C islets were exposed toCXCL12 at concentrations of about 100 ng/ml and about 1 μg/ml or PBSalone and transplanted under the renal capsule of STZ-treated diabeticC57BL/6 recipients. Return to hyperglycemia was considered to be anindicator of graft rejection, while sustained normoglycemia wasconsidered to be an indicator of allograft survival. Coating of isletswith about 1 μg/ml, but not 100 ng/ml, of CXCL12 significantly delayedgraft rejection compared to PBS controls (p=0.012, log-rank test)(12animals per group). FIG. 1B is a set of images characterizing allograftislets. Representative Hematoxylin and Eosin (H&E) staining ofsubcapsular islet graft sites showed reduced mononuclear cellinfiltration in ˜1 μg/ml CXCL12-coated islets compared to uncoatedcontrols (left panels). Insulin staining of ˜1 μg/ml CXCL12 and PBScoated islet graft areas showed a greater number of functional isletsand level of insulin secretion in CXCL12 coated islets compared to PBScontrols (middle panels). Additionally, fluorescent staining showedevidence of CXCL12 staining in CXCL12-coated islets compared to controls(right panels). Costaining of CXCL12 and insulin is shown in a brightershade. FIG. 1C is a bar graph quantifying the number of CD3+ cellspresent in the islet grafts based on CD3 immunostaining, and shows thatthere is significantly decreased infiltration of CD3+ cells into thegraft area in ˜1 μg/ml CXCL12-coated grafts (p=0.001) compared touncoated controls (6 animals per group). FIG. 1D is a bar graphquantifying the number of FoxP3+ cells present in the islet grafts basedon FoxP3 staining, and shows that there is significantly greater FoxP3+cell localization to 1 μg/ml CXCL12-coated grafts than to PBS controls(p=0.0016)(n=6).

FIGS. 2A-2C show that low dose CsA treatment with CXCL12 coatedallogeneic islets does not increase graft survival. FIG. 2A is asurvival curve of the proportion of mice remaining non-diabetic aftertransplantation of CXCL12-coated or uncoated islets and with or withoutCsA treatment. There was a significant reduction in graft survival timewhen CXCL12 coating was combined with CsA treatment at 23 days posttransplantation (p=0.0245, log-rank test). FIG. 2B is a set of images ofimmunohistochemical staining for CD3, FoxP3, and insulin in eachcondition studied. The staining was consistent with survival data shownin FIG. 2A and shows decreased CD3+ and increased FoxP3+ cellinfiltration, as well as greater expression of insulin, in CXCL12 coatedislets compared to CXCL12+ CsA islets. FIG. 2C is a bar graph showingquantification of the number of CD3+ and FoxP3+ cells in islet grafts ineach of the three conditions studied. Significantly decreased numbers ofFoxP3+ cells (p=0.0188) and increased numbers of CD3+ cells (p=0.0002)were observed in CXCL12+ CsA grafts compared to CXCL12 coating alone.

FIGS. 3A-3C show that CXCL12 coating does not affect the number ofC57BL/6-specific CD4 T-Cells. Spleens were removed from C57BL/6 mice 10days post BALB/c islet transplant and splenocytes were stimulated withmitomycin-C treated BALB/c splenocytes in the presence of 1 mM BrdU.Cells were then stained for the incorporation of BrdU. FIG. 3A showsBrdU incorporation in the splenocytes compared to negative stainingcontrol (shaded). FIG. 3B shows that there was no difference in BrdUincorporation into CD4 T-cells from mice which received uncoated (line)and CXCL12-coated islets (shaded). FIG. 3C is a bar graph showing theaverage number of allo-specific CD4 T-cells per spleen in each mousegroup and indicating that coating islets with CXCL12 does not modulatethe systemic immune response to allogeneic tissue. (n=3)

FIGS. 4A-4C show experimental data of CXCL12 coating or PBS exposure ofNOD/LtJ mouse syngeneic islets transplanted under the renal capsule ofSTZ treated diabetic NOD/LtJ mice. FIG. 4A is a survival curve of theproportion of mice remaining non-diabetic after transplantation; thereis a significant difference between CXCL12 and PBS exposed islets(Log-Rank test, p=0.017), indicating prolonged survival and function oftransplanted syngeneic CXCL12 coated islets. FIG. 4B is a bar graphquantifying the number of CD3+ cells in the islet grafts, and shows thatthere was a significant decrease in CD3+ cell infiltration into CXCL12coated islet grafts (p=0.0081). FIG. 4C is a bar graph quantifying thenumber of FoxP3+ cells in the islet grafts, and shows that there wassignificantly increased localization of FoxP3+ cells to CXCL12 coatedislets than to PBS exposed islets (p=0.0019).

FIGS. 5A-5D show experimental data of CXCL12 coating or PBS exposure ofNOD/LtJ mouse syngeneic islets transplanted under the renal capsule ofspontaneously diabetic NOD/LtJ mice. FIG. 5A is a survival curve of theproportion of mice remaining non-diabetic after transplantation; thereis no significant difference between CXCL12 and PBS exposed islets(Log-Rank test, p=0.24). FIG. 5B is a set of images of H&E staining(left panels) showing decreased mononuclear cell infiltration into isletgrafts coated with 1 μg/ml CXCL12 (left panels) and of immunofluorescentstaining for insulin and CXCL12 (right panels) showing increased levelsof both proteins in CXCL12 coated grafts. FIG. 5C is a bar graphquantifying the number of CD3+ cells in the islet grafts, and shows thatalthough there was no difference in survival, there was a significantdecrease in CD3+ cell infiltration into CXCL12 coated islet grafts(p=0.0015). FIG. 5D is a bar graph quantifying the number of FoxP3+cells in the islet grafts, and shows that there was significantlyincreased localization of FoxP3+ cells to CXCL12 coated islets than toPBS exposed islets.

FIGS. 6A-6F show that incorporation of CXCL12 into Ca-LVM alginatecapsules delays rejection of allogeneic and xenogeneic isletstransplanted into the peritoneal cavity. FIG. 6A is a line graph showingkinetics of CXCL12 release from cell-free calcium cross-linked ˜3.3%alginate encapsulant over time in vitro. The concentration of CXCL12 inun-cross-linked sodium alginate was 1 μg/ml; a significant amount ofCXCL12 was lost in the CaCl₂ cross-linking solution (n=3). There were nodifferences in CXCL12 release profiles for alginate concentrations of1.5% to 3.3% (data not shown). Initial release rate of CXCL12 from 1.5%alginate capsules during the first 24 hours was 1.75 ng/ml/hr+/−0.01ng/ml/hr and after four days stabilized at a release rate of 0.18ng/ml/hr+/−0.002 ng/ml/hr. FIG. 6B is a bar graph showing electrostaticinteraction between CXCL12 and barium cross linked alginate capsule. Theleft panel shows that a significantly lower amount of CXCL12 remained inbeads following incubation with 1M NaCl compared to incubation in theabsence of NaCl. The right panel shows that a significantly higheramount of CXCL12 was eluted in the medium following incubation with 1MNaCl compared to incubation in NaCl free medium (n=3, *p<0.05). FIG. 6Cis a bar graph of caspase-3 activity showing that CXCL12 incorporationsignificantly reduces caspase-3 activity in encapsulated murine islets(p=0.0019 for ˜100 ng/ml CXCL12 and p=0.00028 for ˜1 μg/ml CXCL12 versuscontrol). Murine islets were encapsulated with Ca-LVM or Ca-LVMincorporating either ˜100 ng/ml or ˜1 μg/ml CXCL12 (Ca-LVM-CXCL12) andthen cultured in vitro for 48 hours, after which caspase-3 activity wasdetermined. FIG. 6D is a survival plot showing the proportion ofallograft islets after transplantation. Incorporation of 1 μg/ml CXCL12into a Ca-LVM encapsulant delays rejection of allogeneic islets (n=12)for both groups, p=0.0237, Gehan-Breslow-Wilcoxon test). FIG. 6E is asurvival plot of the proportion of allograft islets aftertransplantation. Incorporation of ˜1 μg/ml CXCL12 also delays rejectionof encapsulated allogeneic islets transplanted into allo-sensitizedNOD/LtJ mice (Ca-LVM Capsule, n=7; Ca-LVM-CXCL12 Capsule, n=9; p=0.0066,Gehan-Breslow-Wilcoxon test). FIG. 6F is a survival plot the proportionof allograft islets after transplantation. Incorporation of ˜1 μg/mlCXCL12 significantly delays rejection of encapsulated porcine xenogeneicislets transplanted into diabetic C57BL/6 mice (p=0.0389, log-ranktest). Control and experimental groups: Ca-LVM Capsule, Ca-LVM-10 ng/mlCXCL12 Capsule, Ca-LVM-100 ng/ml CXCL12 Capsule (n=6), Ca-LVM-1 μg/mlCXCL12 Capsule (*p<0.01)(Group size=6)(Gehan-Breslow-Wilcoxon test).

FIGS. 7A-7G show migratory behaviors of T-cell subpopulations to CXCL12and associated CXCR4 expression. FIG. 7A and FIG. 7C are bar graphsshowing migratory responses of CD3+CD8+ T cells. FIG. 7B and FIG. 7D arebar graphs showing migratory response of CD3+CD4+CD25hi T-cells. Thecell migratory responses were quantitated in response to CXCL12, isletscoated with CXCL12 (I-CXCL2) and islets encapsulated withCXCL12(E-CXCL12). CXCL12 was used at a concentration of ˜1 μg/ml in allthree settings. CD8+ and CD4+CD25Hi T-cells underwent low levels ofchemotaxis to ˜1 μg/ml CXCL2 (M/CXCL12) and islets coated orencapsulated with CXCL12. CD8+, but not CD4+CD25Hi, T-cells underwentfugetaxis or chemorepulsion in response to CXCL12 coated or encapsulatedislets Minimal levels of both chemotaxis and fugetaxis were detected forCD8+ or CD4+CD25 Hi T-cells exposed to islets that were not coated withCXCL12 (I-Cont) or encapsulated islets alone (E-Cont).(ns=non-significant; *p<0.05; **p<0.005, Student's t test). To explainthese different migratory responses, CXCR4 expression on CD4+, CD8+, andregulatory T-cells was compared. FIG. 7E shows an example of gatingstrategy for CD8+ and regulatory T-cells. FIG. 7F is a plot showing meanfluorescence intensity (MFI) of CXCR4 expression. Percentage of eachpopulation expressing CXCR4 was calculated, and a representativehistogram in FIG. 7G illustrates the increased expression by Treg cellsof CXCR4 compared to CD4+CD25−and CD8+ T-cells (p<0.0001, Student's ttest).

FIG. 8 is a line graph showing kinetics of CXCL12 retention withincalcium cross-linked 3.3% alginate encapsulant over time in vitro. Theconcentration of CXCL12 in un-cross-linked sodium alginate was 1 μg/ml(n=3). There were no differences in CXCL12 release profiles for alginateconcentrations of 1.5% to 3.3% (data not shown).

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. In case of conflict, thepresent application, including definitions will control.

The term “alginate” as used herein is also known as “alginic acid” andgenerally refers to a carbohydrate polymer (e.g., a polysaccharide)comprising at least two uronate sugars.

The term “allogeneic” means belonging to or obtained from the samespecies.

The term “allograft” refers to a graft of cells or tissue obtained fromthe same species.

The term “xenogeneic” means belonging to or obtained from a differentspecies.

The term “xenograft” refers to a graft of cells or tissue obtained froma different species.

The term “effector T-cell” refers to a differentiated T-cell capable ofmounting a specific immune response by releasing cytokines.

The term “regulatory T-cell” refers to a T-cell that reduces orsuppresses the immune response of B-cells or of other T-cells to anantigen.

By “CXCL12 or SDF-1 polypeptide” is meant a protein or fragment thereofthat binds a CXCL12 specific antibody and that has chemotaxis orfugetaxis activity. Chemotaxis or fugetaxis activity is determined byassaying the direction of T cell migration (e.g., toward or away from anagent of interest). See, for example, Poznansky et al., Nature Medicine2000, 6:543-8.

A “subject” is a vertebrate, including any member of the class mammalia,including humans, domestic and farm animals, and zoo, sports or petanimals, such as mouse, rabbit, pig, sheep, goat, cattle and higherprimates.

As used herein, the terms “treat,”“treating,” “treatment,” and the likerefer to reducing or ameliorating a disorder and/or symptoms associatedtherewith. It will be appreciated that, although not precluded, treatinga disorder or condition does not require that the disorder, condition orsymptoms associated therewith be completely eliminated.

In this disclosure, “comprises,” “comprising,” “containing” and “having”and the like can have the meaning ascribed to them in U.S. patent lawand can mean “includes,” “including,” and the like; “consistingessentially of” or “consists essentially” likewise has the meaningascribed in U.S. patent law and the term is open-ended, allowing for thepresence of more than that which is recited so long as basic or novelcharacteristics of that which is recited is not changed by the presenceof more than that which is recited, but excludes prior art embodiments.

Other definitions appear in context throughout this disclosure.

Compositions and Methods of the Invention

Compositions of the invention are directed to CXCL12 polypeptide elutingmatrices encapsulating at least one cell.

CXCL12 polypeptides are known in the art. See, for example, Poznansky etal., Nature Medicine 2000, 6:543-8. Note that the terms CXCL12 and SDF-1may be used interchangeably. In one embodiment, a CXCL12 polypeptide hasat least about 85%, 90%, 95%, or 100% amino acid sequence identity toNP.001029058 and has chemokine or fugetaxis activity. Exemplary SDF1Isoforms are provided in Table I (below):

TABLE 1 HUMAN SDF1 ISOFORMS  Accession Accession Number Name NumberVersions Sequence SEQ ID NO. SDF-1 NP_954637 NP_954637.1 MNAKVVVVLVSEQ ID NO: 1 Alpha GI:40316924 LVLTALCLSD GKPVSLSYRCPCRFFESHVA TANVKHLKIL NTPNCALQIV ARLKNNNRQV CIDPKLKWIQ EYLEKALNK SDF-1P48061 P48061.1 MNAKVVVVLV SEQ ID NO: 2 Beta GI:1352728LVLTALCLSD GKPVSLSYRC PCRFFESHVA TANVKHLKIL NTPNCALQIVARLKNNNRQV CIDPKLKWIQ EYLEKALNKR FKM SDF-1 NP_001029058 NP_001029058.1MNAKVVVVLV SEQ ID NO: 3 Gamma GI:76563933 LVLTALCLSD GKPVSLSYRCPCRFFESHVA TANVKHLKIL NTPNCALQIV ARLKNNNRQV CIDPKLKWIQ EYEKALNKGRREEKVGKKE KIGKKKRQKK RKAAQKRKN SDF-1 Yu et al. MNAKVVVVLV SEQ ID NO: 4Delta Identification LVLTALCLSD GKPVSLSYRC and expressionPCRFFESHVA TANVKHLKIL of novel NTPNCALQIV isoforms ofARLKNNNRQV CIDPKLKWIQ humna stromal EYEKALNNL ISAAPAGKRV cell-derivedIAGARALHPS PPRACPTARA factor 1. Gene LCEIRLWPPP EWSWPSPGDV(2006) vol. 374 pp.174-9 SDF-1 Yu et al. MNAKVVVVLV SEQ ID NO: 5 EpsilonIdentification LVLTALCLSD GKPVSLSYRC and expressionPCRFFESHVA TANVKHLKIL of novel NTPNCALQIV isoforms ofARLKNNNRQV CIDPKLKWIQ humna stromal EYLEKALNNC cell-derivedfactor 1. Gene (2006) vol. 374 pp.174-9 SDF-1 Yu et al. MNAKVVVVLVSEQ ID NO: 6 Phi Identification LVLTALCLSD GKPVSLSYRC and expressionPCRFFESHVA TANVKHLKIL of novel NTPNCALQIV isoforms ofARLKNNNRQV CIDPKLKWIQ humna stromal EYLEKALNKI WLYGNAETSR cell-derivedfactor 1. Gene (2006) vol. 374 pp.174-9

In another embodiment, the sequence of an exemplary CXCL12/SDF-1polypeptide ismnakvvvvlvlvtalclsdgkpvslsyrcpcrffeshvaranvkhlkilntpncalqivarlknnnrqvcidpklkwiqeylekalnkg rreekvgkkekigkkkrqkkrkaaqkrkn (SEQ ID NO:3).

In yet another embodiment, a CXCL2 polypeptide has at least about 85%,90%, 95%, or 100% amino acid sequence identity to a CXCL12 isoform deltapolypeptide and has chemokine or fugetaxis activity. The sequence of anexemplary CXCL12 isoform delta polypeptideMNAKVVVVLVLVLTALCLSDGKPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQVCIDPKLKWIQEYLEKALNNLISAAPAGKRVIAGARALHPSPPRACPTARALCEIRLWPPP EWSWPSPGDV (SEQ ID NO:4).

CXCL12 polypeptide eluting matrices are characterized, for example, by arelease of the CXCL12 polypeptide at a rate of at least about 1.0ng/mL/hr, e.g., between about 1.0 ng/mL/hr to about 3 ng/mL/hr. Inspecific embodiments, the CXCL12 polypeptide is released at a rate ofabout 1.75 ng/ml/hr. The CXCL12 polypeptide is present in the matrix ata concentration of at least about 100 ng/mL, e.g., between about 100ng/ml to about 1 μg/ml. In specific embodiments, the CXCL12 polypeptideis present in the matrix at a concentration of between about 100 ng/mlto about 1 μg/ml. for about 3 months to about 2 years. Concentrations,release rates and durations will vary according to the selected celltype and disorder to be treated and the selection of appropriateparameters will be known or apparent to those skilled in the art. Ingeneral, the CXCL12 polypeptide is released at a rate sufficient torepel effector T-cells from a specific anatomic site. The ability of aCXCL12 polypeptide eluting matrix to repell effector T-cells can beassessed in vitro, using a boyden chamber assay, as previously describedin Poznansky et al., Journal of clinical investigation, 109, 1101(2002).

Eluting matrices can comprise biocompatible polymers known in the artthat are inert to encapsulated cells (i.e., no stimulation or inhibitionof cell signaling), and are permeable to the CXCL12 polypeptide to beeluted and the molecule to be sensed (e.g. glucose). The matrixthickness is about 200-about 500 microns and in specific embodiments,forms a capsule around the cells. Biocompatible polymers can bebiodegradable or non-degradable. The biocompatible polymer can becarbohydrate-based, protein-based, and/or synthetic, e.g., PLA.Biocompatable materials suitable for use in matrices include, but arenot limited to, poly-dimethyl-siloxane (PDMS), poly-glycerol-sebacate(PGS), polylactic acid (PLA), poly-L-lactic acid (PLLA), poly-D-lacticacid (PDLA), polyglycolide, polyglycolic acid (PGA),polylactide-co-glycolide (PLGA), polydioxanone, polygluconate,polylactic acid-polyethylene oxide copolymers, modified cellulose,collagen, polyhydroxybutyrate, polyhydroxpriopionic acid,polyphosphoester, poly(alpha-hydroxy acid), polycaprolactone,polycarbonates, polyamides, polyanhydrides, polyamino acids,polyorthoesters, polyacetals, polycyanoacrylates, degradable urethanes,aliphatic polyesterspolyacrylates, polymethacrylate, acyl substitutedcellulose acetates, non-degradable polyurethanes, polystyrenes,polyvinyl chloride, polyvinyl flouride, polyvinyl imidazole,chlorosulphonated polyolifins, polyethylene oxide, polyvinyl alcohol,nylon silicon, poly(styrene-block-butadiene), polynorbornene, andhydrogels. Other suitable polymers can be obtained by reference to ThePolymer Handbook, 3rd edition (Wiley, N.Y., 1989). Combinations of thesepolymers may also be used.

In one embodiment, CXCL12 polypeptide eluting matrices of the inventionfurther comprise a secondary layer of cells that express the CXCL12polypeptide, such as mesothelial cells. In other embodiments, the outerlayer of the matrix further comprises an absorbable layer of a CXCL12polypeptide.

In one embodiment, the eluting matrix comprises an alginate (e.g.,alginic acid) and generally refers to a carbohydrate polymer (e.g., apolysaccharide) comprising at least two uronate sugars. The uronatesugars can include, but are not limited to, salts of mannuronic acid (ormannuronate), salts of guluronic acid (or guluronate), and/or isomersthereof. In some embodiments, the alginate can be a linear carbohydratepolymer (e.g., a polysaccharide) comprising mannuronate, guluronateand/or isomers thereof. In some embodiments, alginate can be aco-carbohydrate polymer of mannuronate, guluronate, and/or isomersthereof.

As used herein, the term “isomers” refers to compounds having the samemolecular formula but differing in structure. Isomers which differ onlyin configuration and/or conformation are referred to as “stereoisomers.”The term “isomer” is also used to refer to an enantiomer. The term“enantiomer” is used to describe one of a pair of molecular isomerswhich are mirror images of each other and non-superimposable. Otherterms used to designate or refer to enantiomers include “stereoisomers”(because of the different arrangement or stereochemistry around thechiral center; although all enantiomers are stereoisomers, not allstereoisomers are enantiomers) or “optical isomers” (because of theoptical activity of pure enantiomers, which is the ability of differentpure enantiomers to rotate plane-polarized light in differentdirections). Enantiomers generally have identical physical properties,such as melting points and boiling points, and also have identicalspectroscopic properties. Enantiomers can differ from each other withrespect to their interaction with plane-polarized light and with respectto biological activity. Accordingly, in some embodiments, the salts ofmannuronic acid (or mannuronate) can comprise β-D-mannuronate. In someembodiments, the salts of guluronic acid (or guluronate) can compriseα-L-guluronate.

In some embodiments, alginate can be a block polymer comprising at leastone or more homopolymeric regions of mannuronate (M-blocks), at leastone or more homopolymeric regions of guluronate (G-blocks), at least oneor more regions of alternating structure of mannuronate and guluronate(MG-blocks or GM-blocks).

The proportion, distribution and/or length of these blocks can, in part,determine the chemical and/or physical properties of an alginate gel.For example, the relative content of G and M monomers in the alginatepolymers can affect, e.g., but not limited to, pore size, stability andbiodegradability, gel strength and elasticity of alginate gels. Withoutwishing to be bound by theory, lower G content relative to M content inthe alginate polymers can generally result in more biodegradable gels.Gels with higher 0 content alginate can generally have larger pore sizesand/or stronger gel strength relative to gels with higher M contentalginate, which have smaller pore sizes and lower gel strength. In someembodiments, one or more of the alginate polymers of the alginate matrixcan comprise a M-block content of at least about 10 wt %, at least about20 wt %, at least about 30 wt %, at least about 40 wt %, at least about50 wt %, at least about 60 wt %, at least about 70 wt %, at least about80 wt %, at least about 90 wt % or more. In some embodiments, one ormore of the alginate polymers of the alginate matrix can comprise aG-block content of at least about 10 wt %, at least about 20 wt %, atleast about 30 wt %, at least about 40 wt %/o, at least about 50 wt %,at least about 60 wt %, at least about 70 wt %, at least about 80 wt %,at least about 90 wt % or more. In some embodiments, one or more of thealginate polymers of the alginate matrix can comprise a GM and/orMG-block content of at least 10 wt %, at least about 20 wt %, at leastabout 30 wt %, at least about 40 wt %, at least about 50 wt %, at leastabout 60 wt %, at least about 70 wt %, at least about 80 wt %, at leastabout 90 wt % or more.

In some embodiments, one or more of the alginate polymers of thealginate matrix can comprise a mannuronic acid to guluronic acid (M/G)ratio of about 0.01 to about 100, or about 0.1 to about 50, or about 0.5to about 25, or about 1 to about 20. In some embodiments, one or more ofthe alginate polymers of the alginate matrix can have a M/G ratio ofabout 1 to about 100, or about to about 50, or about 1 to about 25, orabout 1 to about 20, or about 1 to about 10, or about 1 to about 5.

In some embodiments, one or more of the alginate polymers of thealginate matrix can comprise a guluronic acid to mannuronic acid (G/M)ratio of no more than 1.5 or no more than 1. For example, in someembodiments, one or more of the alginate polymers of the alginate matrixcan have a G/M ratio of about 1.5. In some embodiments, one or more ofthe alginate polymers of the alginate matrix can have a G/M ratio ofabout 1. In some embodiments, one or more of the alginate polymers ofthe alginate matrix can have a G/M ratio of less than 1.5, including,e.g., less than 1.4, less than 1.3, less than 1.2, less than 1.1, lessthan 1.0, less than 0.9, less than 0.8, less than 0.7, less than 0.6,less than 0.5, less than 0.4, less than 0.3, less than 0.2, less than0.1, less than 0.05, less than 0.01, less than 0.0075, less than 0.005,less than 0.001 or lower. In some embodiments, one or more of thealginate polymers of the alginate matrix can have a G/M ratio of lessthan 1, including, e.g., less than 0.9, less than 0.8, less than 0.7,less than 0.6, less than 0.5, less than 0.4, less than 0.3, less than0.2, less than 0.1, less than 0.05, less than 0.01, less than 0.0075,less than 0.005, less than 0.001, less than 0.0001, or lower.

In some embodiments, one or more of the alginate polymers of thealginate matrix can comprise a guluronic acid to mannuronic acid (G/M)ratio of at least about 1.5 or higher. For example, in some embodiments,one or more of the alginate polymers of the alginate matrix can have aG/M ratio of about 1.5. In some embodiments, one or more of the alginatepolymers of the alginate matrix can have a G/M ratio of greater than1.5, including, e.g., greater than 2, greater than 2.5, greater than 3,greater than 3.5, greater than 4, greater than 4.5, greater than 5,greater than 6, greater than 7, greater than 8, greater than 9, greaterthan 10, greater than 15, greater than 20, greater than 30, greater than40, greater than 50, greater than 60, greater than 70, greater than 80,greater than 90, greater than 100 or higher.

The average molecular weight of alginate polymers can affect, e.g.,gelling time, pore size, gel strength and/or elasticity of gels.Alginate polymers can have average molecular weights ranging from about2 kDa to 10000 kDa. Without wishing to be bound by theory, lowermolecular weight of the alginate polymer can generally result in morebiodegradable gels. In some embodiments, the alginate polymers of thealginate matrix can have an average molecular weight of about 5 kDa toabout 10,000 kDa, or about 10 kDa to about 5000 kDa, or about 25 kDa toabout 2500 kDa, or about 50 kDa to about 1000 kDa, or about 50 kDa toabout 500 kDa, or about 50 kDa to about 250 kDa. In some embodiments,the alginate polymers of the alginate matrix can have an averagemolecule weight of about 5 kDa to about 350 kDa. In some embodiments,the alginate polymers of the alginate matrix can have an averagemolecule weight of about 2 kDa to about 100 kDa. In some embodiments,the alginate polymers of the alginate matrix have an average moleculeweight of about 50 kDa to about 500 kDa. In some embodiments, thealginate polymers of the alginate matrix have an average molecule weightof about 50 kDa to about 300 kDa. In some embodiments, the alginatepolymers of the alginate matrix have an average molecule weight of about75 kDa to about 200 kDa. In some embodiments, the alginate polymers ofthe alginate matrix have an average molecule weight of about 75 kDa toabout 150 kDa. In some embodiments, the alginate polymers of thealginate matrix have an average molecule weight of about 150 kDa toabout 250 kDa. In some embodiments, the alginate polymers of thealginate matrix have an average molecule weight of about 100 kDa toabout 1000 kDa.

In some embodiments, the alginate polymers of the alginate matrix canhave an average molecular weight of less than 75 kDa or lower. In someembodiments, the alginate polymers of the alginate matrix can have anaverage molecular weight of at least about 75 kDa, at least about 80kDa, at least about 90 kDa, at least about 100 kDa, at least about 110kDa, at least about 120 kDa, at least about 130 kDa, at least about 140kDa, at least about 150 kDa, at least about 160 kDa, at least about 170kDa, at least about 180 kDa, at least about 190 kDa, at least about 200kDa, at least about 250 kDa, at least about 300 kDa, or higher.

In one embodiment, the alginate polymers of the alginate matrix has anaverage molecular weight of about 75 kDa to about 200 kDa, with aguluronic acid to mannuronic acid (G/M) ratio of about 1. In oneembodiment, the alginate polymers of the alginate matrix has an averagemolecular weight of about 75 kDa to about 200 kDa, with a guluronic acidto mannuronic acid (G/M) ratio of less than 1.

Without limitations, the molecular weight can be the peak averagemolecular weight (Mp), the number average molecular weight (Mn), or theweight average molecular weight (Mw).

The alginate can be derived from any source and/or produced by anyart-recognized methods. In some embodiments, the alginate can be derivedfrom stem and/or leaves of seaweeds or kelp. In some embodiments, thealginate can be derived from green algae (Chlorophyta), brown algae(Phaeophyta), red algae (Rhodophyta), or any combinations thereof.Examples of seaweeds or kelps include, but are not limited to, varioustypes of Laminaria (e.g., but not limited to, Laminaria hyperborea,Laminaria digitata, and Laminaria japonica), Lessonia nigrescens,Lessonia trabeculata, Durvillaea antarctica. Ecklonia maxima,Macrocystis pyrifera, Ascophyllum nodosum, and any combinations thereof.

In some embodiments, the alginate can be a bacterial alginate, e.g.,produced by a microbial fermentation using bacteria. Examples ofbacteria that can be used in alginate production include, but are notlimited to, Pseudomonas (e.g., Pseudomonas Aeruginosa) and Azotobacter(e.g., Azobacter Vinelandii). In some embodiments, the bacteria canproduce a polysaccharide polymer with a structure resembling alginate,for example, differing in that there are acetyl groups on a portion ofthe C2 and C3 hydroxyls.

In some embodiments, the alginate can be modified. In some embodiments,the alginate can be chemically modified. For example, a chemicallymodified alginate can comprise propylene glycol alginate (PGA). In someembodiments, PGA can be made by contacting a partially neutralizedalginic acid with propylene oxide gas under pressure. The propyleneoxide can react exothermically with the alginic acid to form a mixedprimary/secondary ester.

In some embodiments, the alginate can be of clinical grade, e.g.,suitable for use in vivo. In some embodiments, the alginate can bepurified prior to use for cell encapsulation. See, e.g., Mallet andKorbutt, Tissue Eng Part A. 2009. 15(6):1301-1309. In some embodiments,the alginate can have low endotoxin. For example, endotoxins can bepresent in the alginate in an amount of no more than 150 EU/gram, nomore than 100 EU/gram, no more than 75 EU/gram, no more than 50 EU/gram,no more than 25 EU/gram, no more than 20 EU/gram, no more than 10EU/gram, no more than 5 EU/gram, no more than 1 EU/gram, no more than0.5 EU/gram, no more than 0.1 EU/gram.

Any art-recognized alginate can be used in the methods of variousaspects described herein. Examples of alginates that can be used in thecompositions described herein include, without limitations, sodiumalginate (sodium salt of alginic acid), potassium alginate (potassiumsalt of alginic acid), calcium alginate, magnesium alginate,triethanolamine alginate, PGA, and any combinations thereof. In someembodiments, soluble alginate can be in the form of mono-valent saltsincluding, without limitation, sodium alginate, potassium alginate andammonium alginate. In some embodiments, the alginate can be calciumalginate. In one embodiment, calcium alginate can be made from sodiumalginate from which the sodium salt has been removed and replaced withcalcium. Alginates described in and/or produced by the methods describedin the International Patent Application Nos. WO 2007/140312; WO2006/051421; WO2006/132661; and WO1991/007951 and U.S. Pat. No.8,481,695 can also be used in the compositions and methods of variousaspects described herein. In some embodiments, commercially-availablealginates, e.g., obtained from FMC BioPolymer and Novamatrix, can alsobe in the compositions and methods of various aspects described herein.

Alginate generally forms a gel matrix in the presence of divalent ionsand/or trivalent ions. Non-limiting examples of divalent or trivalentions that can be used to form alginate gels include calcium ions, bariumions, strontium ions, copper ions, zinc ions, magnesium ions, manganeseions, cobalt ions, lead ions, iron ions, aluminum ions, and anycombinations thereof.

In some embodiments, the alginate matrix can be covalently crosslinked.Examples of covalent crosslinking agents that can be used to covalentlycrosslink alginate include, but are not limited to, carbodiimides, allylhalide oxides, dialdehydes, diamines, and diisocyanates.

Eluting matrix formulations of the invention include those suitable forinjection, infusion or implantation (subcutaneous, intravenous,intramuscular, intraperitoneal, intradermal, parenteral, rectal, and/orintravaginal or the like), inhalation, oral/nasal or topicaladministration. The formulations may conveniently be presented in unitdosage form and may be prepared by any methods well known in the art ofpharmacy. The amount of CXCL12 polypeptide which can be combined in adosage form will vary depending upon the host being treated, theparticular mode of administration, e.g., injection or implantation.Formulations of this invention can be prepared according to any methodknown to the art for the manufacture of pharmaceuticals and can containsweetening agents, flavoring agents, coloring agents and preservingagents. A formulation can be admixtured with nontoxic pharmaceuticallyacceptable excipients which are suitable for manufacture. Formulationsmay comprise one or more diluents, emulsifiers, preservatives, buffers,excipients, etc. and may be provided in such forms as liquids,emulsions, creams, lotions, gels, on patches and in implants.

CXCL12 polypeptide eluting matrices of the invention encapsulate atleast one cell. Encapsulated cells can include, but are not limited to,stem cells, neuronal cells, smooth or skeletal muscle cells, myocytes,fibroblasts, chondrocytes, adipocytes, fibromyoblasts, ectodermal cells,including ductile and skin cells, hepatocytcs, kidney cells, livercells, cardiac cells, pancreatic cells, islet cells, cells present inthe intestine, osteoblasts and other cells forming bone or cartilage,and hematopoietic cells. In specific embodiments, the cell is an insulinproducing cell, such as an islet cell (e.g., a porcine islet cell, ahuman islet cell or an islet cell derived from a stem or iPS cell).

Eluting matrices of the invention are refillable CXCL12 polypeptidedelivery devices implanted or otherwise inserted within a patient. Forexample, the matrix may comprise a needle or catheter entry port so thatcells can be infused or removed without removing the matrix from thepatient. Alternatively, the eluting matrices can be repeatedlyadministered to a subject (e.g., a “sensitized subject”), withoutassociated immune rejection.

CXCL12 polypeptide eluting matrices of the invention are useful in thetreatment of autoimmune diseases including, but not limited to,rheumatoid arthritis, uveitis, insulin-dependent diabetes mellitus,hemolytic anemias, rheumatic fever, Crohn's disease, Guillain-Barresyndrome, psoriasis, thyroiditis, Graves' disease, myasthenia gravis,glomerulonephritis, autoimmune hepatitis, and systemic lupuserythematosus.

In one embodiment, CXCL12 polypeptide eluting matrices of the inventioncan be formulated with islet cells for use in the treatment of diabetes.Diabetes is a condition in which a person has a high blood sugar(glucose) level as a result of the body either not producing enoughinsulin, or because body cells do not properly respond to the insulinthat is produced. In healthy persons, blood glucose levels aremaintained within a narrow range, primarily by the actions of thehormone insulin. Insulin is released by pancreatic beta-cells at anappropriate rate in response to circulating glucose concentrations, theresponse being modulated by other factors including other circulatingnutrients, islet innervation and incretin hormones. Insulin maintainsglucose concentrations by constraining the rate of hepatic glucoserelease to match the rate of glucose clearance.

Insulin thus enables body cells to absorb glucose, to turn into energy.If the body cells do not absorb the glucose, the glucose accumulates inthe blood (hyperglycemia), leading to various potential medicalcomplications. Accordingly, diabetes is characterized by increased bloodglucose resulting in secondary complications such as cardiovasculardiseases, kidney failure, retinopathy and neuropathy if not properlycontrolled.

Two major pathophysiologies are related to increase glycemia. The firstis an autoimmune attack against the pancreatic insulin-producingbeta-cells (Type 1 diabetes) whilst the second is associated to poorbeta-cell function and increased peripheral insulin resistance (Type 2diabetes). Similar to Type 1, beta-cell death is also observed in Type 2diabetes. Type 1 and often Type 2 diabetes requires the person to injectinsulin.

Type 1 DM is typically characterized by loss of the insulin-producingbeta-cells of the islets of Langerhans in the pancreas leading toinsulin deficiency. This type of diabetes can be further classified asimmune-mediated or idiopathic. The majority of Type 1 diabetes is of theimmune-mediated nature, where beta-cell loss is a T-cell mediatedautoimmune attack. Type 2 DM is characterized by beta-cell dysfunctionin combination with insulin resistance. The defective responsiveness ofbody tissues to insulin is believed to involve the insulin receptor.Similar to Type 1 diabetes an insufficient beta cell mass is also apathogenic factor in many Type 2 diabetic patients. In the early stageof Type 2 diabetes, hyperglycemia can be reversed by a variety ofmeasures and medications that improve insulin secretion and reduceglucose production by the liver. As the disease progresses, theimpairment of insulin secretion occurs, and therapeutic replacement ofinsulin may sometimes become necessary in certain patients.

Regulatory T-cells are a subset of CD4+ T cells originated from thethymus, which are generally known to play a significant role inmaintenance of tolerance. Regulatory T-cells actively play a role inimmune modulation, and suppress alloimmune responses of transplantrejection (C. A. Piccirillo. Cytokine 43, 395 (Sep., 2008); G. Xia etal. Translational research: the journal of laboratory and clinicalmedicine 153, 60 (Feb., 2009); K. J. Wood. Transplantation proceedings43, 2135 (July-August, 2011); and G. Feng et al. Transplantation 86, 578(Aug. 27, 2008)). Regulatory T-cells prevent murine autoimmune diabetesand control autoreactive destruction of transplanted islets (M. J.Richer et al. PloS one 7, e31153 (2012) and D. R. Tonkin et al. Immunol181, 4516 (Oct. 1, 2008)). Islet transplantation represents apotentially curative approach to diabetes, however, in previous studiesof islet transplantation, systemic immune suppression could not achievelong-term control of blood glucose levels due to immune-mediatedrejection of transplanted islets. Incorporation of CXCL12 into a matrixencapsulating transplanted islets provides both a physical and abiological barrier to cell-mediated and humoral anti-islet immunity. TheCXCL12 polypeptide repels effector T-cells and recruitsimmune-suppressive regulatory T-cells, while reducing or eliminating theneed for systemic immunosuppression. Accordingly, in one embodiment,CXCL12 polypeptide eluting matrices of the invention are useful for theregeneration, replacement or substitution (partial or wholly) of atleast part of the pancreas of a patient deficient in pancreatic cells,particularly beta-cells without concomitant immunosuppression. Anypatient whose pancreas does not produce sufficient insulin, or indeedany insulin, may benefit from such therapy. Insufficient insulinproduction includes the production of lower levels of insulin comparedto a normal (healthy) subject, but it also includes subjects who produceinsulin levels that are comparable to normal (healthy) subjects but whorequire higher insulin levels, for example due to insulin resistance,excessive food consumption, morbid obesity and the like.

CXCL12 polypeptide eluting matrices of the invention selectively recruitregulatory T-cells, thereby prolonging survival of the implanted matrixand providing protection from immune destruction in even a sensitizedhost. Accordingly, CXCL12 polypeptide eluting matrices of the inventionare retrievable, and can be repeatedly administered, if desired, withoutimmune system rejection. Furthermore, CXCL12 polypeptide elutingmatrices of the invention provide sustained islet survival andcontinuous production of CXCL12 polypeptides from the encapsulatedislets, for at least about 1 month to about 2 years. During this time,the fasting serum concentration of glucose in the subject is maintainedat a blood level of between about 80 mg/dl and about 120 mg/dl.According, CXCL12 polypeptide eluting matrices of the invention areparticularly useful in the treatment of diabetes.

In a specific embodiment, a CXCL12 polypeptide eluting matrix for use intreating diabetes comprises about 1.5 to about 2% w/v of a highmannuronic acid, calcium cross-linked alginate, about 100 ng/ml to about1 μg/ml of a CXCL12 polypeptide and at least one islet cell, wherein theCXCL12 polypeptide is released at a rate of between about 1.0 ng/ml/hrto about 3 ng/ml/hr. An exemplary encapsulation procedure begins withpreparation of the islet cells, by mixing donor islets with 2 mLfiltered dithizone/PBS. 80 mg of alginate-LVM (Pronova UP LVM SodiumAlginate) is mixed in 5 ml, 300 mOsmo NaCl until dissolved. About 800 toabout 1000 islets are spun down in cRPM for 3 minutes at 300 rpm×G,re-suspended in DMEM and spun again at 300 rpm×G for 3 minutes. A 1 mL,syringe can be used to mix 0.75 mL alginate with islets. Alginate andislets can be loaded into a 60 mL syringe using 18 g needle. The loadedsyringe is placed into the encapsulator, having the voltage set at themaximum (e.g., 1.21 kV), the frequency set at 1500 and the amplitude setat maximum. After encapsulation, stir capsules for about 5 minutes in300 mOsmo CaCl2 and filter capsules into fresh beaker, wash and culturewith DMEM.

The present invention is additionally described by way of the followingillustrative, non-limiting Examples that provide a better understandingof the present invention and of its many advantages.

EXAMPLES

The following examples illustrate some embodiments and aspects of theinvention. It will be apparent to those skilled in the relevant art thatvarious modifications, additions, substitutions, and the like can beperformed without altering the spirit or scope of the invention, andsuch modifications and variations are encompassed within the scope ofthe invention as defined in the claims which follow. The followingexamples do not in any way limit the invention.

Islet transplantation represents a potentially curative approach to TypeI Diabetes. However, the islet transplantation generally requiressystemic immune suppression to control immune-mediated rejection oftransplanted islets and there is a limited human islet supply. Thefollowing Examples show that the chemokine, CXCL12, can repel effectorT-cells while recruiting immune-suppressive regulatory T-cells (Tregs)to an anatomic site, and coating or encapsulating donor islets withCXCL12 can induce local immune-isolation and protect an allo- orxenograft without systemic immune suppression. In the followingExamples, islet transplantation was performed in murine models ofinsulin-dependent diabetes. Coating of islets with CXCL12 ormicroencapsulation of islets with alginate incorporating the chemokine,CXCL12, resulted in prolonged allo- and xenoislet survival and function,as well as a selective increase in Treg infiltration. These data asshown below indicate the use of CXCL12 as coating or a component ofalginate encapsulant to induce local immune-isolation for allo- orxenoislet transplantation while abrogating the need for systemicimmunosuppression.

Example 1 Direct Coating of Alloislets with CXCL2 Polypeptide

In this Example, it was sought to determine whether coating alloisletswith CXCL12 polypeptides prior to transplantation could result inprolonged islet survival and function as well as accumulation ofregulatory T cells (Tregs) at the graft site. The islet capsulegenerally contains fibronectin, and, without wishing to be bound bytheory, CXCL12 can both stably bind to and elute from this matrixprotein (15). Accordingly, islets from BALB/C mice were exposed to abuffered solution (e.g., PBS) or coated with CXCL12 at a concentrationof about 100 ng/ml or about 1 μg/ml and transplanted under the leftkidney capsule of streptozotocin (STZ)-treated diabetic C57BL/6 mice.Mice were sacrificed at the point when they returned to a diabetic statewith two sequential blood glucose recordings of >250 mg/dl. Allogeneicislet grafts coated with CXCL12 at ˜1 μg/ml resulted in the maintenanceof recipient mice in a non-diabetic state for a significantly longertime than alloislets that were exposed to PBS alone (p=0.012;Kaplan-Mayer log rank test) (FIG. 1A). Islets coated with 100 ng/ml ofCXCL12 polypeptides were rejected at a similar rate to PBS-exposedislets (p=0.31). Mononuclear cell infiltration into the grafts wasreduced in the context of CXCL12 coating at about 1 μg/ml compared toPBS controls (FIG. 1B). Insulin expression and CXCL12 presence were alsoshown in CXCL12-coated alloislets compared to PBS-treated allografts(FIG. 1B). CD3 and FoxP3 T-cell infiltration into grafts was quantified.CD3+ T-cell infiltration into islet grafts was significantly reduced inCXCL12 compared to PBS-exposed alloislets as determined byimmunohistochemistry (FIG. 1C, p=0.001). Further, CXCL12 coating ofislet allografts was associated with a significant increase in FoxP3+T-cell infiltration within and around the CXCL12-coated graft comparedto PBS-exposed alloislets (FIG. 1D, p=0.0016).

Example 2 Concurrent Use of CXCL12 Coating and Low Dose Cyclosporine A

It was next sought to determine whether concurrent use of systemicimmunosuppression, for example, in the form of low dose of Cyclosporin A(CsA)(e.g., ˜2 mg/kg) treatment, could enhance CXCL12-coated alloisletsurvival. While CXCL12 coating alone was shown to prolong alloisletsurvival compared to PBS-exposed controls in these experiments(p=0.0245), CXCL12 coating in combination with low dose CsA treatmentdid not appear to extend islet survival and the combination of CXCL12coating and CsA treatment could result in reduced islet survival incomparison to CXCL12 coating alone (p=0.046) (FIG. 2A). Significantdifferences in islet survival and between control and experimentalgroups were shown by 23 days post transplantation. Staining of isletgrafts showed heavy infiltration of CD3+ T-cells in CXCL12 coating plusCsA treated animals compared to CXCL12 coating alone (p=0.0002) or CsAtreated alone (p=0.0027) (FIGS. 2B-2C). The addition of low dose CsA tothe treatment of animals receiving CXCL12-coated islets also led to asignificant reduction in FoxP3 cell infiltration into the graft comparedto CXCL12 coated islets alone (p=0.0188)(FIG. 2C). It was previouslydiscussed that CsA inhibits specific elements of the signaling pathwayfor CXCL12, and, for example, chemokine-mediated cell migration (16).The findings presented herein indicate that concurrent use of CXCL12coating and low dose cyclosporine A does not appear to augment an immuneprotective effect in alloislet transplantation.

Since the CXCL12-coated islets show a longer survival than PBS-exposedislets, it was next sought to determine whether this phenomenon was dueto an effect of the chemokine on cell-mediated anti-islet immunity. Toascertain the level of allo-reactivity, a mixed lymphocyte reaction(MLR) was carried out from mice 21 days after they received eitherCXCL12-coated or uncoated islet transplants. No difference was detectedbetween these two groups, indicating that CXCL12 creates an anatomicniche that can slow T-cell-mediated rejection but does not impact thegeneration of anti-islet immunity (FIGS. 3A-3C). Thus, without wishingto be bound by theory, in some embodiments, CXCL12 coating can inducelocal immune isolation rather than preventing the generation of systemicanti-allogeneic cell-mediated responses.

Example 3 Transplantation of CXCL12-coated Syngeneic Islets inPrediabetic and Diabetic Mouse Model

To determine whether CXCL12 coating could play a role in reducing orpreventing rejection in the context of syngeneic islet transplantation,diabetic NOD/LtJ mice was used. In this model, syngeneic islets fromnon-diabetic NOD/LtJ mice were transplanted into STZ-treated diabeticNOD/LtJ mice. CXCL12 coating of syngeneic islets led to a significantlylonger period of normoglycemia than PBS-exposed islets (FIG. 4A)(p=0.017). Histopathological and immunohistochemical studies showedheavy mononuclear cell infiltration into PBS-treated islets but notCXCL12-coated islets (images not shown). That is, H&E staining showeddecreased mononuclear cell infiltration into islet grafts coated with ˜1μg/ml CXCL12 and immunofluorescent staining for insulin and CXCL12showed increased levels of both proteins in CXCL12 coated grafts.Staining for CXCL12 and insulin in this experiment showed healthyinsulin-producing and CXCL12-positive islets in comparison toPBS-exposed islets. CXCL12 coating of syngeneic islets also reduced CD3+T-cell infiltration into donor islets compared to PBS-exposed syngeneicNOD/LtJ islets (FIG. 4B, p=0.0081). CXCL12 coating of syngeneic NOD/LtJislets also led to a significantly increased number of FoxP3+ cells inthe islet graft site compared to PBS-treated controls (p=0.0019) (FIG.4C). While CXCL12 coating of syngeneic islets did not appear to reducethe rate of recurrence of diabetes in spontaneously diabetic NOD/LtJmice (FIG. 5A) (p=0.24), hematoxylin and eosin and insulin/CXCL12staining showed reduced mononuclear cell infiltration and increasedinsulin expression in CXCL12-coated islets compared to controls (FIGS.5B-5C). In addition, there was a consistent reduction in CD3+ T-cellinfiltration and increased FoxP3+ cell infiltration into theCXCL12-coated graft compared to PBS controls (FIG. 5D). Taken together,CXCL12 coating of syngeneic islets can prevent rejection in aprediabetic model but not necessarily in a diabetic model. This lack ofa pro-survival effect of CXCL12 coating in this syngeneic transplantsetting can be attributable to, e.g., inefficiency of blockingestablished humoral anti-islet immune responses by a CXCL12 coating oftransplanted islets.

Example 4 Use of Alginate Encapsulant (Islet Cells Encapsulated inAlginate) Incorporating CXCL12 in Sensitized and Non SensitizedRecipients

It was previously discussed that early expression of insulinautoantibodies correlates with progression to diabetes and probablyprimarily reflects insulitis (17-19). As shown in Example 3, theefficacy of CXCL12 coating of islets for transplantation was moreeffective as an intervention in the setting of pre-formed anti-isletantibodies. In this Example, it was sought to determine if incorporationof CXCL12 into alginate microcapsules could protect transplanted isletsby providing both a physical and a biological barrier to cell-mediatedand humoral anti-islet immunity.

In one embodiment, the encapsulation matrix comprised of 2% lowviscosity, high mannuronic acid, calcium cross-linked alginate (Ca-LVM)and CXCL12 polypeptides incorporated therein (referred to as“Ca-LVM-CXCL12” hereafter). FIG. 6A shows that Ca-LVM-CXCL12encapsulants resulted in prolonged release of CXCL12 in vitro at about1.75+/−0.4 ng/ml/hr after an initial rapid release over the first 3hours. There was also prolonged retention of CXCL12 in the alginatematrix, resulting in residual intracapsular CXCL12 concentration between100 and 200 ng/ml after 22 days of in vitro incubation of cell-freecapsules (data not shown). Without wishing to be bound by theory, theprolonged retention of CXCL12 in the matrix was likely due toelectrostatic interactions between the positively charged chemokine (pIof 9) and the negatively charged alginate (pI of 2) (20). This wassupported by the observation that CXCL12 was effectively eluted into themedium as a result of incubation with 1M NaCl in comparison to mediumcontaining no NaCl. Similarly, CXCL12 was retained within the capsulewhen incubated in the absence of NaCl but was extracted from the capsulein 1M NaCl. (FIG. 6B). As CXCL12 has also been shown to be apro-survival factor for islets, the effect of the incorporation ofCXCL12 into a Ca-LVM encapsulant on islet viability was also evaluated.The findings presented herein show that incorporation of CXCL12 into theencapsulant significantly decreased the level of caspase-3 activity inencapsulated islets determined at least after 48 hours of in vitroculture as compared to unmodified Ca-LVM alginate (100 ng/ml CXCL12;p=0.0019)(1 μg/ml CXCXL12; p=0.00028) (FIG. 6C).

Alloislet transplantation of Ca-LVM-CXCL12 encapsulants intospontaneously diabetic NOD/LtJ mice without systemic immune suppressionwas evaluated. When alloislets were transplanted into the peritonealcavity of diabetic NOD/LtJ mice, incorporation of CXCL12 into the Ca-LVMalginate significantly prolonged islet function and survival as comparedto the unmodified Ca-LVM alginate (Mean days in non diabetic statepost-transplantation —Ca-LVM-CXCL12=136; Ca-LVM=62) (p=0.048) (FIG. 6D).Histopathological studies of retrieved Ca-LVM-CXCL12 capsules at 12weeks post-transplant showed intact islet morphology in comparison tounmodified Ca-LVM capsules in which islets were necrotic or degenerative(Data not shown). Islets encapsulated with Ca-LVM-CXCL12 appeared viableand intact using phase contrast microscopy and H&E staining at 6 weekspost-transplantation in comparison to necrotic islets encapsulated inunmodified Ca-LVM. Accordingly, CXCL12 incorporation improves islethealth and decreases necrosis six weeks after transplant. Alloisletsfrom C57BL/6 mice were then transplanted into spontaneously diabeticNOD/LtJ mice that had previously received and rejected a skin transplantfrom C57/B6 mice. Alloislets encapsulated with Ca-LVM-CXCL12 survivedand maintained a normoglycemic state significantly longer in recipientsthan islets encapsulated with Ca-LVM alone (FIG. 6E). This shows thatCXCL12 incorporation into encapsulant protected islets from an immunememory response in recipient mice.

It was then sought to determine whether xenoislets could be protected byan alginate encapsulant that incorporated CXCL12. Porcine xenoisletswere encapsulated in Ca-LVM or Ca-LVM with CXCL12 at a concentration ofabout 10 ng/ml, about 100 ng/ml or about 1 μg/ml, and transplanted intothe peritoneal cavity of diabetic C57BL/6 mice. Porcine isletsencapsulated with Ca-LVM containing CXCL12 at a concentration of about 1μg/ml sustained normoglycemia in recipient mice for a significantlylonger period of time than either Ca-LVM or Ca-LVM-CXCL12 (˜10 ng/ml)encapsulated islets (FIG. 6F).

Example 5 Effect of CXCL12 Coating or Microencapsulation on CD8+ T-cellsand Treg Cells in vitro

In order to determine whether the mechanism by which CXCL12 coating orincorporation in the encapsulant sustains immune isolation of the xenoor alloislet graft involves selective repulsion of CD8+ T-cells andattraction of CD4+ Treg cells to the graft site, the expression of CXCR4on and migration of T-cell subpopulations from NOD/LtJ mice were studiedby flow cytometry and transmigration assays, respectively. T-cellsderived from NOD/LtJ mice in response to medium alone, recombinantCXCL12, CXCL12-coated or Ca-LVM-CXL12 encapsulated islets were studiedin Boyden chamber based assays. Upper chambers were loaded with purifiedCD3+CD8+ T cells (FIGS. 7A and 7C) or CD4+CD25hi+ Treg cells (FIGS. 7Band 7D) for each condition. Upper and lower wells were loaded withmedia, CXCL12 (˜1 μg/ml) or islets coated with CXCL12 (˜1 μg/ml) orencapsulated with Ca-LVM-CXCL12 (˜1 μg/ml). Both CD3+CD8+ T-cells andCD4+CD25Hi Tregs underwent chemotaxis in response to CXCL12,CXCL12-coated or Ca-LVM-CXCL12-encapsulated islets. However, asignificantly larger fugetactic response was detected when CD8+ cellswere incubated with CXCL12, CXCL12-coated islets orCa-LVM-CXCL12-encapsulated islets than Treg cells. No detectable levelsof Treg cell fugetaxis were measured in the conditions performed.

It was next sought to determine whether the differential migratoryresponses to CXCL12 between CD8+ T cells and CD4+ Treg cells could be,in part, due to the differential expression of the chemokine's cognatereceptor, CXCR4 on these two T-cell subpopulations. CD8+ T-cells fromthe spleen of NOD/LtJ mice expressed significantly lower levels of CXCR4than CD4+CD25HiFoxP3+ Treg cells (p<0.005)(FIGS. 7E and 7F). These datashow that CXCL12 coating or CXCL12 incorporation in an encapsulantsurrounding transplanted alloislets can result in preferentialrecruitment of Treg cells within the graft while repelling CD8+ T-cellsin this transplant model.

Discussion

The findings presented in Examples 1-5 show that that coating of isletswith CXCL12 can lead to a delay in islet rejection in allo and insyngeneic islet transplantation in STZ treated diabetic NOD/LtJ mice.Specifically, it was observed that a Ca-LVM based alginate encapsulantincorporating CXCL12 protected alloislets with both pre-existent humoraland cell mediated anti-islet responses in the context of transplantationinto diabetic and sensitized NOD/LtJ mice.

The transplant models in Examples 1-5 show that coating or encapsulationof islets with CXCL12 results in accumulation of Treg cells at the graftsite.

Accordingly, without wishing to be bound by theory, in both tumor andtransplant settings the retention/accumulation of Treg cells can beassociated with the establishment of an immune suppressivemicroenvironment. The findings presented herein show that coating orencapsulation of allo or xenoislets with CXCL12 can lead to delayedislet rejection and concomitant prolonged islet function through theinduction of local immune isolation. The discovery that CXCL12incorporation into clinical grade alginate encapsulant surroundingtransplanted allo and xenoislets allows sustained islet function andprotection from immune destruction in the sensitized host withoutsystemic immune suppression is a surprising and also clinicallytranslatable finding.

Exemplary Materials and Methods Used in Examples 1-5

Animals and Induction of Diabetes. Six-week-old female BALB/c (H2^(d)),6-week-old female C57BL/6 (H2^(b)), and 6-8 week old female NOD/LtJ(H2^(g7)) mice were used. Animals can be purchased, e.g., from JacksonLaboratory (Bar Harbor, Me.). All procedures were carried out followingthe Public Health Service Policy on Humane Care of Laboratory Animalsand approved by the Subcommittee on Research Animal Care atMassachusetts General Hospital. Hyperglycemia was spontaneous in18-20-week-old NOD/LtJ mice and was induced in 4- and 6-week-old NOD/LtJand 6-week-old C57BL/6 mice by intraperitoneal (IP) injection of 200mg/kg streptozotocin (Sigma-Aldrich, St. Louis, Mo.). Mice with threeconsecutive blood glucose readings above 250 mg/di were consideredhyperglycemic.

Pancreatic Islet Isolation, CXCL12 Coating and Incorporation of theChemokine into Alginate Encapsulant. Primary islets were isolated fromdonor mice as previously described in Papeta et al., Transplantation 83,174 (2007). For example, islets were isolated from female 6-week-oldBALB/c donors, female 6-week-old C57BL/6 donors, or female 4-week-oldNOD/LtJ donors. Pancreata were infused via the common bile duct withLiberase TL (e.g., ˜83 μg/ml) (Roche Diagnostics, Indianapolis, Ind.)and digested for ˜20 minutes at ˜37° C. Islets were purified on apolysucrose/glucose density gradient (Mediatech, Manassas, Va.) andselected under a microscope. The islets were cultured in RPMI 1640(Mediatech, supplemented with 10% fetal bovine serum, 1%Penicillin/Streptomycin, and 1% L-Glutamine) for at least 2 days orlonger to allow for recovery. A minimum of 450 islets to be transplantedvia the kidney capsule were incubated at ˜37° C. for ˜3 hours prior totransplantation in DPBS (Mediatech) with or without ˜100 ng/ml or ˜1μg/ml CXCL12 (PeproTech Inc, Rocky Hill, N.J.). Separately, prior toencapsulation, CXCL12 was incorporated into liquid Na-LVM alginate(ultra-pure LVM, Novamatrix) at a concentration of ˜1 μg/ml.

Production and Transplantation of Ca-LVM Alginate Capsules.Approximately 1000 C57BL/6 islets were mixed in ˜0.75 mL of ˜1.5%alginate (ultra-pure LVM, Novamatrix, Drammen, Norway)) in ˜300 mOsmoNaCl solution with or without ˜1 μg/ml CXCL12. The mixture was then runthrough a syringe driven encapsulator (Inotech Research Encapsulator,IE-50R) using a 300 μm nozzle charged to 1.21 kV vibrating at 1500 Hz.Alginate was cross-linked, e.g., in 300 mOsmo (approximately 118 mM)CaCl₂, for about 5 minutes, filtered, and washed with DMEM (Mediatech)to remove excess calcium.

CXCL12 Retention and Release from Alginate Capsules and Caspase-3 Assay.Prior to capsule formation, ˜3.6% LVM was mixed with ˜10 μg/mL CXCL12(Peprotech, Rocky Hill, N.J.) stock solution to yield ˜3.3% LVM with ˜1μg/mL CXCL12. Calcium cross-linked LVM capsules were formed using anelectrostatic droplet generator (Nisco Engineering, Zurich,Switzerland), at a charge of 5 kV, using a 0.5 mm nozzle and a flow rateof 30 mL/h. The CXCL12-containing acellular capsules were incubated innon tissue culture treated multi-well plates in DMEM (Sigma Aldrich, St.Louis, Mo.) with ˜1.6 g/L bovine serum albumin (Sigma Aldrich). Theratio of capsules to medium was maintained at about 1:2 v/v throughoutthe course of the experiment. At each time point, a ˜0.1 mL sample ofcapsules was removed and solubilized using a 110 mM sodium citratesolution. Additionally, at each time point, all medium was removed andfresh medium was added. Both medium and solubilized capsule samples wereassayed for CXCL12 content by ELISA (R&D Systems, Minneapolis, Minn.).In order to determine the effect of encapsulation in combination withCXCL12 on islet apoptosis, murine islets were encapsulated with alginateencapsulant as described herein with or without ˜1 μg/ml CXCL12. As acontrol, islets can also be exposed to streptozotocin, a known inducerof islet apoptosis. For example, islets can be cultured in thiscondition in vitro for 48 hours and Caspase-3 activity in encapsulatedislets determined using an ELISA based assay (R and D systems,Minneapolis, Minn.).

CXCL12 Salting Out from Alginate Capsules. CXCL12 capsules were formedas described above with the following modification; capsules werecross-linked using 20 mM calcium chloride to prevent their dissolutionin a high concentration sodium chloride solution. Capsules wereincubated in ˜1M NaCl with ˜0.1% BSA for ˜6 hours, after which thesolution was collected and the beads were solubilized using 200 mMethylenediaminetetraacetic acid disodium salt (ED2SS), pH 9.0. Thesolution and solubilized capsule samples were assayed for CXCL12 contentby ELISA, as described above. Control capsules were incubated in NaCLfree CaCl₂ solution and CXCL12 retained and released from the capsulesdetermined.

Islet Transplantation Models. Variably treated islets were transplantedunder the left renal capsule of recipient mice or were encapsulated inCa-LVM alginate and were transplanted via the peritoneal cavity. Fourdifferent exemplary transplant models were used: (1) BALB/C islets weretransplanted under the renal capsule of STZ-induced diabetic C57BL/6mice; (2) Pre-diabetic (pre-sensitized) NOD/LtJ islets were transplantedunder the renal capsule of spontaneously diabetic NOD/LtJ mice; (3)Pre-diabetic (pre-sensitized) NOD/LtJ islets were transplanted under therenal capsule of 6-week-old diabetic (pre-sensitized, STZ-induceddiabetic) NOD/LtJ mice; and (4) C57BL/6 islets were encapsulated inCa-LVM alginate and transplanted into the peritoneal cavity of 6 weekold STZ-induced diabetic NOD/LtJ mice.

Monitoring Recipients' Glycemic Control. Recipients' tail vein bloodglucose level was monitored at least twice a week and was used tointerpret islet graft function. Graft rejection was defined as a returnto hyperglycemia (e.g., characterized by three consecutive measurementsabove 250 mg/dl). Mice were sacrificed upon graft rejection.

Immunohistochemistry and Immunofluorescence Staining. Kidneys containingsub-capsular islet grafts were fixed in 4% formaldehyde, embedded inparaffin, and 5 μm sections of grafts were cut for slides. Some sectionswere stained with hematoxylin and eosin (H&E). Immunohistochemistry wasalso performed using primary antibodies for CD3 (Dako, Denmark), FoxP3(eBioscience, CA), and insulin (Dako, Denmark). An appropriate secondaryantibody was then paired to each primary antibody and DAB(3,3′-Diaminobenzidine) was used as a substrate for the staining.Nuclear staining (blue) was performed with Meyer's haemalum for theavidin-biotin complex IHC staining method. Camera images were taken witha Zeiss Axio Observer Z1 at 10× magnification and islet graft sectionswere manually scored for CD3⁺ and/or FoxP3⁺ T-cell infiltration. Thenumbers of cells positive for each marker were recorded in 5 randomlyselected high-power fields near the graft site. Immunofluorescentstaining was also performed using fluorescent primary antibodies forinsulin (guinea pig anti-insulin, 1:800, Invitrogen) and CXCL12 (rabbitanti-mouse CXCL12, 1:200, Cell Sciences). Slides were washed, incubatedwith the primary antibodies overnight at 4° C. and then incubated withthe appropriate secondary antibodies for one hour at room temperature(goat anti-guinea pig IgG [Dylight 488, 1:200, Jackson Immunoresearch]and goat anti-rabbit IgG [Dylight 549, 1:200, Jackson Immunoresearch]).Following secondary antibody application, tissues were washed, counterstained with To-Pro-3 (1:5000, Invitrogen), and mounted. Digital imagesof immunofluorescence slides were obtained by means of confocalmicroscopy (LSM 5 Pascal, Carl Zeiss).

Cyclosporin A Treatment. Diabetic C57BL/6 mice were transplanted with450 BALB/c islets coated with PBS or CXCL12 (˜1 μg/ml). Recipients weretreated with one of three treatment groups: (A) Cyclosporin A (CsA,Sigma) injection for recipients of PBS-coated islets; (B) CsA injectionfor recipients of CXCL12-coated islets; and (C) PBS injection forrecipients of CXCL12-coated islets. For groups A and B, CsA was preparedby dissolving in ethanol and injected subcutaneously at ˜2 mg/kg on theday of transplantation and daily, thereafter, until graft rejection.

Mixed Lymphocyte Reaction. C57BL/6 mice (n=3/group) for responder celldonation were allocated to one of three groups: CXCL12-coated islettransplant, non-coated islet transplant, or no treatment. 10 days aftervariable treatment, spleens were mechanically disrupted and filtered,and red blood cells were lyzed using M-lyse buffer (R&D Systems).Allogeneic stimulator cells from untreated BALB/c mouse spleens weresimilarly prepared and incubated with 20 μg/ml Mitomycin C (SigmaAldrich) for 1 hour at 37° C., washed repeatedly with medium, andco-cultured at 37° C. and 5% CO₂ with responder cells for three days ata 1:1 ratio. Eighteen hours prior to the completion of the stimulation,the cells were pulsed with a 1 mM solution of bromodeoxyuridine (BrdU,BD Biosciences). After 72 hours of culture, cells were washed andsubsequently stained with anti-mouse CD3 (APC-Cy7), CD8 (PerCP), and CD4(PE-Cy7) (BD Biosciences). Cells were permeabilized withCytofix/Cytoperm (BD Biosciences), subjected to DNase digestion (SigmaAldrich), and stained with PE-conjugated anti-BrdU antibody (BDBiosciences). Flow cytometric analysis was performed on a LSRII (BDBiosciences) and data analyzed with FlowJo software (TreeStar) for Tcells per spleen that were stimulated and that incorporated BrdU minusthe number of unstimulated cells per spleen that incorporated BrdU.

Transmigration Assays. Two types of migration assays were performed.First, cell migration was measured using a Boyden Chamber (96-wellformat, 3-μm pore; ChemoTx System, Neuro Probe Inc, Gaithersburg, Mass.)as previously described in Poznansky et al., Journal of clinicalinvestigation, 109, 1101 (2002). For example, ˜30 μL of RPMI 1640supplemented with 0.5% FBS, along with 50 control islets or isletscoated with 1 μg/ml CXCL12 (PeproTech Inc, Rocky Hill, N.J.), was addedto the lower and upper chambers. 7,000 T-reg cells or CD8 T-cells,isolated with the CD4, CD25 and/or CD8 MACS separation kits,respectively (Miltenyi Biotec, Auburn, Calif.), were loaded into theupper chamber of the Boyden Chamber. After a three-hour incubation at37° C. and 5% CO₂, cells on the upper surface of the membrane wereremoved and migrated cells in the lower chamber were counted intriplicate. Separately, migration to encapsulated islets, with orwithout CXCL12 incorporated into the capsules, was also measured using aTranswell system (24-well format, 3 μm pore, Corning). Briefly, 500 μlof RPMI 1640 supplemented with 0.5% FBS, alone or with 10 control or ˜1μg/ml CXCL12-incorporated capsules, was loaded into the bottom chamber.100 μl of media, alone or with variably treated capsules, was loadedinto the upper chamber along with 10⁵ cells of the desired population.Cells were incubated for 3 hours at 37° C. and 5% CO₂ and the number ofmigrated cells was counted in triplicate. For both assays, atransmigration index was calculated as the ratio between the number ofcells counted in the presence of CXCL12 and in media-only controls.

Quantitation of CXCR4 Expression on T-cell Subsets. Splenocytes wereharvested from untreated NOD/LtJ mice and red blood cells were lysedusing Mouse Erythrocyte Lysing Buffer (R&D Systems). Cells were thenstained for the surface markers CD3ε, CD4, CD8α, CD25, and CXCR4(CD184)(CD3ε: clone 145-2C11; CD4: clone RM 4-5; CD8α: clone 53-6.7; CD25:clone PC61; CXCR4: clone 2B11, BD Biosciences), fixed, permeabilizedwith BD Cytofix/Cytoperm (BD Biosciences) according to themanufacturer's instructions, and stained for the intracellular markerFoxP3 (clone MF23, BD Biosciences). Flow cytometry was performed on a 4Laser LSR II (BD Biosciences). Data were analyzed using FlowJo software(Tree Star) and gating was accomplished using a fluorescence-minus-onegating strategy.

Statistical Analysis. Islet graft survival between groups was comparedusing the Kaplan-Meier method, and the survival data were analyzed byusing the GraphPad Prism 5 statistic software. Numerical variables werecompared using Student's t test. A p-value below 0.05 was consideredstatistically significant.

From the foregoing description, it will be apparent that variations andmodifications may be made to the invention described herein to adopt itto various usages and conditions. Such embodiments are also within thescope of the following claims. The recitation of a listing of elementsin any definition of a variable herein includes definitions of thatvariable as any single element or combination (or subcombination) oflisted elements. The recitation of an embodiment herein includes thatembodiment as any single embodiment or in combination with any otherembodiments or portions thereof.

REFERENCES

All patents, patent applications and publications mentioned in thisspecification are herein incorporated by reference to the same extent asif each independent patent and publication was specifically andindividually indicated to be incorporated by reference. Incorporation byreference herein includes, but is not limited to:

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We claim:
 1. An eluting matrix suitable for implantation into a mammal,said matrix comprising at least one protein expressing-cell, whereinsaid cell is encapsulated in a CXCL12-eluting porous polymeric-matrixwhich is permeable to the protein, and further wherein the elution rateof CXCL12 from the matrix is from about 1 ng/mL/hr to about 3 ng/mL/hrso as to repel effector T-cells surrounding said matrix for a period ofat least one month after implantation.
 2. The eluting matrix of claim 1,wherein the elution rate of CXCL12 is about 1.75 ng/mL/hr.
 3. Theeluting matrix of claim 1, wherein the CXCL12 is present in the matrixat a concentration of at least about 100 ng/mL.
 4. The eluting matrix ofclaim 1, wherein the CXCL12 is present in the matrix at a concentrationof between about 100 ng/mL to about 1 μg/mL.
 5. The eluting matrix ofclaim 4, wherein the CXCL12 is present in the matrix at a concentrationof between about 100 ng/mL to about 1 μg/mL for about 3 months to about2 years after implantation.
 6. The eluting matrix of claim 1, whereinthe matrix has a thickness of from about 200 microns to about 500microns.
 7. The eluting matrix of claim 1, further comprising asecondary layer of cells that express a CXCL12 polypeptide.
 8. Theeluting matrix of claim 1, further comprising an outer layer of a CXCL12polypeptide.
 9. The eluting matrix of claim 1, wherein said at least oneprotein expressing-cell is selected from the group consisting of amyocyte, a fibroblast, a chondrocyte, an adipocyte, a fibromyoblast, anectodermal cell, a kidney cell, a liver cell, a pancreatic cell, anintestinal cell, an osteoblast, and a hematopoietic cell.
 10. Theeluting matrix of claim 1, wherein said at least one cell is selectedfrom the group consisting of a neuronal cell, a smooth muscle cell, askeletal muscle cell, a cardiac cell, an epithelial cell, and ahepatocyte.
 11. The eluting matrices of claim 1, wherein said at leastone cell is a stem cell.
 12. An eluting matrix suitable for implantationinto a mammal, said matrix comprising at least one xenogenic islet cellcapable of expressing insulin, wherein said cell is encapsulated in aCXCL12-eluting porous alginate matrix having a thickness of from about200 microns to about 500 microns which is permeable to the insulin, andfurther wherein the elution rate of CXCL12 from the matrix is from about1 ng/mL/hr to about 3 ng/mL/hr so as to repel effector T-cellssurrounding said matrix which is maintained for a period of at least onemonth after implantation.
 13. The eluting matrix of claim 12, whereinthe elution rate of CXCL12 is about 1.75 ng/mL/hr.
 14. The elutingmatrix of claim 12, wherein the CXCL12 is present in the matrix at aconcentration of at least about 100 ng/mL.
 15. The eluting matrix ofclaim 12, wherein the CXCL12 is present in the matrix at a concentrationof between about 100 ng/mL to about 1 μg/mL.
 16. The eluting matrix ofclaim 12, wherein the CXCL12 is present in the matrix at a concentrationof between about 100 ng/mL, to about 1 μg/mL for about 3 months to about2 years after implantation.
 17. The eluting matrix of claim 12, whereinsaid alginate matrix is covalently crosslinked.
 18. The eluting matrixof claim 12, wherein said matrix comprises about 1.5 to about 2% w/v ofa high mannuronic acid, calcium cross-linked alginate.
 19. The elutingmatrix of claim 12, wherein said alginate matrix is comprised ofalginate polymer subunits having an average molecular weight of lessthan 75 kDa.
 20. The eluting matrix of claim 12, wherein said alginatematrix is comprised of alginate polymer subunits having an averagemolecular weight of about 75 kDa to about 200 kDa.
 21. The elutingmatrix of claim 12, wherein said alginate matrix is comprised ofmannuronic acid and guluronic acid.
 22. The eluting matrix of claim 21,wherein said alginate matrix comprises a mannuronic acid to guluronicacid ratio of about 1:100 to about 100:1.
 23. The eluting matrix ofclaim 21, wherein said alginate matrix comprises a guluronic acid tomannuronic acid ratio of no more than 3:2.